Major histocompatibility complex (MHC) class I molecules bind endogenously synthesized peptides for presentation to cytotoxic T cells. The human class I molecule HLA-B27 consists of a trimolecular complex containing the HLA-B27 heavy chain, a peptide that is usually nine amino acid residues (aa) long, and beta2-microglobulin (beta2m). The key interactions for peptide selectivity are between Glu-45, which forms a salt bridge with the Arg at P2 of the peptide and Asp-116 which favors the binding of peptides containing a Lys or Arg at P9. The t1/2 of dissociation of [125I]beta2m was measured for peptide-specific HLA-B27 wild-type (wt) and mutant complexes. HLA-B27 wt and HLA-B27D116F formed relatively stable complexes, with a t1/2 of dissociation on the scale of hours, with appropriate peptides that contained Arg at P2, whereas HLA- B27 E45T required a Gln at P2. Similarly, kinetically stable D116F complexes were formed only with peptides that contained a Leu or Val at P9 instead of Arg or Lys. The [125I]beta2m dissociation rate data were fit to a set of equations in order to calculate relative binding coefficients for each anchor residue at P2 and P9. The P2 coefficients were sensitive only to the D116F mutation. Thus, drastic structural changes in one subsite do not affect the other subsite, indicating that the dominant anchor residues at P2 and P9 independently contribute to stabilizing the class I/peptide complex. Antigenic peptides are presented to CD4+ T cells by MHC class II molecules via a highly polymorphic peptide-binding groove. The two HLA- DR alleles isotopically expressed on HLA-DR15Dw2-positive cells, DRB1*1501 (DR2b) and DRB5*0101 (DR2a) molecules, show a number of differences in polymorphic residues of the beta-chain, including a Gly- Val-dimorphism at position beta86. Therefore, different requirements for interaction of peptides with the alleles are expected. In this study, naturally processed self-peptides were eluted from purified HLA-DR15Dw2 molecules and related to DRB1*1501 or DRB5*0101 molecules by binding assays. An alignment of self-peptides and foreign peptides allowed the delineation of putative anchor motifs. DRB5*0101 requires a bulky hydrophobic residue (F or Y) at position i as a primary anchor, and Q or an aliphatic residue, such as V,I, or M, at position i +3; positively charged residues at positions i + 7 and i + 8 are secondary anchors. For DRB1*1501, a nonaromatic, hydrophobic anchor (L,V, or I) at position i is supplemented by a bulky hydrophobic residue (F or Y) at position i + 3 as a primary anchor; an additional hydrophobic side chain represented by M, I, V, or F occurs at position i + 6. Because HLA-DR15 Dw2 is associated with susceptibility to develop multiple sclerosis, the delineation of ligand motifs of the two DR2 alleles may help to study the interaction between potential autoantigenic peptides and these molecules in the future.